Aminoacylation of tRNAs by their cognate aminoacyl tRNA synthetases is fundamental to protein synthesis. This reaction attaches an amino acid to the 3xe2x80x2 end of a tRNA so that the amino acid can be delivered to the growing polypeptide chain as the anticodon sequence of the tRNA reads a codon triplet in a mRNA. The specificity of aminoacylation is determined by the ability of an aminoacyl tRNA synthetase to interact with the correct amino acid and to recognize its cognate tRNA through specific nucleotides (Schimmel, P. and Sxc3x6ll, D. (1979) Ann. Rev. Biochem. 48, 601-648; Meinnel et al. (1995) in tRNA: Structure, biosynthesis, and function, Sxc3x6ll, D. and RajBhandary, U., eds, pages 251-292, American Society for Microbiology, Washington, DC). Interference with either the amino acid binding step or the tRNA recognition step of a synthetase can inhibit aminoacylation and arrest protein synthesis, thereby leading to cell death. tRNA also functions as a primer for reverse transcriptase by viral polymerase during the replication cycle of retroviruses.
Several amino acid analogs have proven useful as inhibitors of aminoacylation (Aldridge, K. E. (1992) Antimicrobial Agents and Chemotherapy 36, 851-853; Yanagisawa et al. (1994) J. Biol. Chem. 269, 24303-24309). However, these analogs are not targeted at the tRNA recognition step.
Recent studies have shown that the specific nucleotides important for tRNA recognition can vary from one organism to another (Sampson et al. (1989) Science 243, 1363-1366; Nazarenko et al. (1992) Nucleic Acids Res. 20, 475-478). The species-specific nucleotides account for the species-selective aminoacylation of tRNA acceptor stems by the cognate synthetases (Hipps et al. (1995) Proc. Natl. Acad. Sci. USA 92, 5550-5552; Hou et al. (1995) RNA 1, 707-713; Quinn et al. (1995) Biochemistry 34, 12489-12495). Substitutions of these nucleotides reduce aminoacylation while transfer of these nucleotides to the analogous positions of a heterologous tRNA (tRNA from a different organism) confer recognition and aminoacylation of that tRNA by the heterologous synthetase.
However, targeting specific nucleotides of tRNAs has proven difficult. Previous studies demonstrated that the secondary cloverleaf structure of tRNAs and its L shaped tertiary structure limited the accessibility of complementary oligoribonucleotides to specific regions (Uhlenbeck, O. C. (1972) J. Mol. Biol. 65, 25-41; Schimmel et al., (1972) Biochemistry 11, 642-646; Freier, S. M. and Tinoco, I. (1975) Biochemistry 14, 3310-3314) These accessible regions include the NCCA sequence at the 3xe2x80x2 end, the anticodon loop, a portion of the D loop, and a portion of the variable loop. None of the nucleotides in the stem regions were accessible to oligoribonucleotides. In those early studies, oligoribonucleotides of 3-4 residues in length were used. In a more recent study, a 13-mer oligoribonucleotide was used to probe the anticodon loop of E. coli tRNAfMet (Hayase et al. (1990) Biochemistry 29, 8793-8797). However, due to the unusual conformation of the anticodon loop, the 13-mer did not gain access to the tRNA until the higher order tRNA structure was melted first.
It has now been found that modified oligonucleotides can target species-specific tRNA nucleotides and provide a basis for drug design for inhibition of activities of a tRNA from one organism but not from others. In the present invention, oligonucleotide sequences which bind to the critical nucleotides in a tRNA that are recognized by the cognate aminoacyl tRNA synthetase are provided. Such oligonucleotides are useful as antibiotics, inhibiting aminoacylation of a tRNA of a selected pathogen and thereby killing the pathogen. Using the methods of the present invention, oligonucleotides which inhibit other activities of pathogenic tRNA can also be designed.
An object of the present invention is to provide a method of inhibiting activities of a tRNA of a selected pathogen comprising contacting a cell infected with the selected pathogen with an effective amount of an oligonucleotide targeted to a region of a cloverleaf structure of the tRNA of the selected pathogen.
Another object of the present invention is to provide compositions capable of inhibiting activity of a tRNA of a selected pathogen comprising an oligonucleotide targeted to a region of a cloverleaf structure of the tRNA of the selected pathogen.